Giardia lamblia is a ubiquitous human pathogen and a Category B bioterrorism agent that causes mal-absorptive diarrhea. One of the most compelling yet understudied aspects of Giardia biology is its cytoskeleton: microfilaments are responsible for the ability of Giardia to mechanically attach to intestinal epithelial cells thus permitting the parasite to establish infections. This R21 proposal will explore this new area of research by initiating identification of novel proteins of the microfilament cytoskeleton and establishing the foundations of biophysical understanding of the mechanism by which attachment is controlled. The following hypotheses will be tested: (1) The microfilament cytoskeleton in Giardia will be significantly more divergent than previously recognized thus establishing it as an important drug target. Novel actin-binding proteins will be identified through biochemical, genetic and proteomics means, including affinity chromatography, sedimentation, yeast two-hybrid, and mass spectrometry. (2) Attachment of Giardia to biological and inert substrates will be mediated by integrated contraction of the microfilaments under the dorsal surface of the plasma membrane (to generate negative pressure and a suction force under the ventral surface of the cell) and of microfilaments around the perimeter of the ventral disk (to generate a clutching force). The force of attachment will be quantified for the first time using flow cells to create hydrodynamic shear forces. The mechanism of attachment will be determined by observing the dynamics and force of attachment to flexible or perforated substrates and by using total interference reflection aqueous-fluorescence (TIRAF) microscopy to quantify the distance between the parasite ventral surface and substrate. The studies proposed here will lay the groundwork for a more detailed study of the mechanism by which Giardia uses microfilaments to generate attachment force. This work will advance our understanding of parasite pathogenesis and develop our knowledge of microfilaments as a target for drug design.